T lymphocytes isolated from whole blood are utilized in a wide variety of in vitro, in vivo, and clinical research and therapeutic applications. Examples include studies of immune response, T cell receptor signaling, cytokine release and gene expression profiling. Perhaps most significantly, isolation and subsequent ex vivo engineering of T lymphocytes for subsequent transplantation into clinical patients is showing tremendous promise as a novel cancer therapy. The main approaches to this are engineering of T cells to express either chimeric antigen receptors (CAR) or T cell receptors (TCR). In both approaches, T cells are isolated from whole blood, activated and expanded ex vivo, and subsequently infused into human subjects.
Although both polyclonal and antigen-specific T cells can be readily isolated from whole blood, their numbers are limited. Accordingly, protocols that activate and promote ex vivo expansion of T cells are widely used. Such ex vivo manipulations, however, can potentially reduce T cell viability, proliferation, and survival after infusion. Thus, the choice of methods used for T cell activation has important implications for clinical efficacy.
It is well-established that, in vivo, activation of T cells is dependent on two signals; engagement of the T cell receptor with antigen (signal 1) and ligation of a costimulatory molecule (signal 2). Both are required for an effective immune response. Ex vivo, T cell activation is most commonly induced by exposing the T cells to antibodies directed against the T cell surface markers CD3 and CD28 to engage the T cell receptor and deliver a costimulatory signal simultaneously.
There are significant disadvantages of conventional ex-vivo T cell activation protocols, which use magnetic beads, resulting from the presence of residual magnetic beads attached to the cells. These may negatively affect both function and viability. Pre-clinical clinical applications require cells are that are free from contaminating particles, while retaining high viablilty. For example, June et al. (Pilot study of redirected autologous T cells engineered to contain humanized anti-CD19 in patients with relapsed or refractory CD19+ leukemia and lymphoma previously treated with cell therapy (2015) ClinicalTrials.gov) specified final product release criteria in the IND included the specifications that the number of anti-CD3/CD28-coated paramagnetic beads should not exceed 100 per 3×106 cells and that cell viability should be greater than 70%. However, minimizing the number of beads represents a formidable obstacle in the clinical translation of such therapies, as most antibody-coated magnetic-bead based products lack the ability to readily release bound cells from capture molecules in a manner that does not alter the viability and phenotype of the isolated cells.
Given the significant interest in, and rapid expansion of, T cell engineering-based cancer therapies, there is a significant need for improved T cell activation and harvesting methods that overcome the above limitations of existing approaches, particularly for downstream clinical applications. Specifically, there is a substantial need for technologies to enable ex-vivo cell expansion protocols to meet clinical specifications, to consistently and reproducibly activate T cells, to preserve cell viability and function, and to be applicable to different cell sources and activating agents.